Designed ankyrin repeat protein libraries (WO2002/020565; Binz, H. K., Amstutz, P., Kohl, A., Stumpp, M. T., Briand, C., Forrer, P., Grütter, M. G., and Plückthun, A., Nat. Biotechnol. 22, 575-582, 2004; Stumpp, M. T., Binz, H. K and Amstutz, P., Drug Discov. Today 13, 695-701, 2008) can be used for the selection of target specific designed ankyrin repeat domains. Such target specific designed ankyrin repeat domains in turn can be used as valuable components of recombinant binding proteins for the treatment of diseases. The selection of different designed ankyrin repeat domains with binding specificity for human epidermal growth factor receptor 2 (HER2 or ErbB2; UniProt P04626) has been described (WO2014/083208). In the present invention recombinant binding proteins are disclosed comprising designed ankyrin repeat domains with binding specificity for HER2. Unlike e.g. IgG antibodies, which exhibit long systemic half-lives mediated by FcRn recycling, proteins comprising designed ankyrin repeat domains typically exhibit a fast pharmacokinetic clearance and short terminal half-lives, unless the protein comprises elements that improve the pharmacokinetic properties, such as e.g. a designed ankyrin repeat domain with binding specificity to serum albumin described in WO2012/069654. Using serum albumin binding for improving pharmacokinetic properties of proteins is a process well-known in the art (see e.g. WO1991/001743; Frejd F. Y., 2012 (in Kontermann, R (Ed.) “Therapeutic proteins: strategies to modulate their plasma half-lives”, Wiley-VCH Verlag GmbH, 2012, ISBN 978-3-527-32849-9); and WO2012/069654). In order to use designed ankyrin repeat domains with binding specificity for serum albumin in clinical drug candidates, it is desirable that the storage stability of known designed ankyrin repeat domains with binding specificity for serum albumin is improved. Disclosed herein are recombinant binding proteins comprising designed ankyrin repeat domains with binding specificity for serum albumin, wherein said designed ankyrin repeat domains with binding specificity for serum albumin exhibit improved storage stability properties. In contrast to earlier reports (Hopp, J., Horning, N., Zettlitz, K. A., Schwarz, A., Fuss, N., Müller, D., Kontermann, R. E. Protein Eng. Des. Sel. 23, 827-834, 2010), we surprisingly observed that by having two designed ankyrin repeat domains with binding specificity for serum albumin instead of one in the recombinant binding protein, the pharmacokinetic properties of the recombinant binding protein can be improved.
HER2 plays an important role in the pathogenesis and progression of certain types of cancer. HER2 is a trans-membrane receptor tyrosine kinase (RTK) belonging to the wider family of ErbB receptors (Bublil, E. M. and Yarden, Y. Curr. Opin. Cell Biol. 19(2), 124-34, 2007). The ErbB receptor family is conserved across vertebrates and also includes ErbB1 or epidermal growth factor receptor (EGFR) or HER1 (UniProt P00533) and the receptors HER3 (ErbB3; UniProt P21860) and HER4 (ErbB4; UniProt Q15303). All ErbB receptors share extensive sequence and domain homologies, and form functional homodimers (e.g. ErbB1-ErbB1, HER2-HER2 and HER4-HER4) and heterodimers in all combinations. Receptor homo- and heterodimerization occurs upon ligand binding or receptor overexpression, and in turn activates intracellular receptor kinase domains by autophosphorylation. This then triggers downstream intracellular signaling and biological responses. Well-known antagonists of the ErbB signaling pathways include the monoclonal antibodies Trastuzumab (binding to domain IV of the extracellular domain of HER2 and inhibiting HER2 homodimerization) and Pertuzumab (binding to domain II of the extracellular domain of HER2 and inhibiting HER2/HER3 heterodimerization). Importantly, Trastuzumab has mainly an anti-proliferative effect and tumors may escape form such treatment in advanced disease stages. Pertuzumab, which has an unexpectedly low therapeutic efficacy as a single agent, can complement the activity of Trastuzumab by interfering with the HER2/HER3 heterodimerization. Thus, the combination of Trastuzumab with Pertuzumab is attractive for the treatment of HER2 positive cancer (Capelan M., et al., Ann. Oncol., 24, 273-82, 2013).
The combination of Trastuzumab and Pertuzumab has led to the concept that dual targeting of two domains in HER2 is required for superior anti-tumor efficacy. Antibody mixtures targeting domains II and IV of HER2, or simultaneous targeting of domain I and another domain of HER2 (US 20110033460; e.g. also domain IV), or domain I and domain IV (WO2014/060365; Jost, Ch., et al., Structure 21, 1-13, 2013; Tamaskovic, R., et al., Nat Commun 7, 11672, 2016), or domain II and domain IV (WO2014/083208), or simultaneous targeting of the Trastuzumab epitope on domain IV of HER2 and the Pertuzumab epitope on domain II of HER2 (WO2009/068625) have been reported. Some of the approaches included bi-paratopic binding proteins. Interestingly, some of the bi-paratopic binding proteins tested in WO2009/068625 had antagonistic effect, others agonistic effects. Reports of WO2014/083208 and Jost, Ch., et al (loc. cit.; WO2014/060365) indicate that the generation of antagonistic bi-paratopic binding proteins is not straight forward. Instead, careful selection of the individual domains (epitope, binder properties) as well as the structural arrangement (orientation, distance, linker length, linker composition) have to be optimized for effective antagonism. Additionally, the choice of the pharmacokinetic engineering moiety and its structural arrangement also have to be optimized. Together, this represents a choice from at least 250,000 different variants. Disclosed herein is a recombinant binding protein comprising (i) a bi-paratopic binding protein antagonizing ErbB-signaling consisting of two designed ankyrin repeat domains with binding specificity for HER2 and (ii) two designed ankyrin repeat domains with binding specificity for serum albumin and with improved storage stability. This recombinant binding protein, a DARPin® drug candidate, is shown to be a valuable drug candidate for the treatment of various diseases. DARPin® is a registered trademark of Molecular Partners AG, Switzerland.